Mycoplasma alligatorisInfection Promotes CD95 (FasR) Expression and Apoptosis of Primary Cardiac Fibroblasts

Abstract

Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. A genome survey implicated sialidase and hyaluronidase, potential promoters of CD95-mediated eukaryotic cell death, as virulence factors of M. alligatoris. We used immunofluorescence imaging and flow cytometry to examine the effects of M. alligatoris infection in vitro on CD95 expression and apoptosis by alligator cardiac fibroblasts, a major cell type of a target organ of M. alligatoris infection in vivo. A uniform distribution of CD95 in primary cultured cardiac, skeletal muscle, and embryonic fibroblasts was demonstrated by using polyclonal antibodies against the N or C terminus of mouse or human CD95. Anti-CD95 antibodies reacted on Western blots of fibroblast lysates with a band with the predicted apparent molecular weight of CD95, but soluble CD95 was not detected in plasma from control or M. alligatoris-infected alligators. The proportion of CD95-gated cardiac fibroblasts increased threefold (P < 0.01) 48 h after inoculation with M. alligatoris. Infection induced morphological changes in cardiac fibroblasts, including translocation of CD95 characteristic of apoptosis and an eightfold increase (P < 0.16) in 5-bromo-2-deoxyuridine (BrdU) incorporation measured in a terminal deoxynucleotide transferase dUTP nick end-labeling apoptosis assay. The proportion of BrdU-gated controls activated with agonistic immunoglobulin M against human CD95 also increased threefold (P < 0.03 for muscle). Heat-inactivated M. alligatoris and sterile M. alligatoris-conditioned culture supernatant had no effect. This is the first report of a CD95 homolog in the class Reptilia and establishes a new model that can be used to test the direct bacterial interaction with upstream components of the CD95 signal transduction pathway.In contrast to the usually subtle mycoplasmoses, Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts (7). Pathological changes in affected individuals reflect multisystem inflammatory disease, including necrotic epicarditis, pericarditis, and myocarditis (8, 9). A comparative genome survey approach to the identification of candidate virulence mechanisms revealed that M. alligatoris strain A21JP2 T possesses genes for the "spreading factors" sialidase (nanI) and hyaluronidase (nagH), a combination unprecedented among mycoplasmas (10). Functional studies demonstrated expression of the corresponding glycosidase activities, which might degrade host extracellular matrix (ECM) glycans to facilitate the rapid invasiveness of M. alligatoris infection (16,28,31,41,48). Its attenuated sibling species, Mycoplasma crocodyli strain MP145 T (43), also possesses hyaluronidase, so direct ECM damage alone seems insufficient to explain the particular virulence of M. alligatoris. M. crocodyli MP145 T does not possess sialidase (10). Desialylation of the eukaryotic cell death inducer CD95 (the fibroblast-associated receptor FasR) by sialidase substantially promoted CD95-mediated apoptosis in B-lineage leukemias and Jurk...

Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis

“…Subconfluent (50-70%) fibroblasts were inoculated with approximately 10 9 CFU of M. alligatoris (initial multiplicity of infection approximately 10 4 ) and incubated as described (Hunt and Brown, 2005). To test the effect of sialidase inhibition, infected cultures were coincubated with 50 or 500 μg/ml DANA.…”

“…The fluorescence intensity, measured by flow cytometry, was proportional to the total number of all activated caspase 1, 3, 4, 5, 6, 7, 8, and 9 molecules. Apoptosis was detected by immunofluorescence staining and a species-independent terminal deoxynucleotide transferase dUTP nick end-labeling (TUNEL) assay (APO-BrdU TUNEL Assay Kit; Molecular Probes) as described (Hunt and Brown, 2005). Apoptosis was induced in positive controls by activation with an agonistic IgM mAb against the extracellular domain of CD95 (CH11; Upstate, Lake Placid NY) as described for alligator cardiac fibroblasts (Hunt and Brown, 2005).…”

“…alligatoris strain A21JP2 T was cultured as described (Hunt and Brown, 2005). Sialidase activity was assayed quantitatively in 96-well plates (FluoroNunc 437112; Nalge Nunc International, Rochester NY) using the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUAN; Sigma-Aldrich, St. Louis MO) and a spectrofluorometer (SpectraMax M5 with SoftMax Pro 5 software; Molecular Devices, Sunnyvale CA).…”

“…IgG1 (AHS4411 [SFF-2]; BioSource International, Camarillo CA) and ; Abcam) mAbs against the standard isoform of human CD44, and monoclonal ; American Type Culture Collection) and polyclonal IgG1 (AHS4441; BioSource International) antibodies which recognize epitopes encoded by exons v3-v10 of human variant CD44 isoforms, were also tested. CD95 expression was detected by fluorescence imaging and flow cytometry as described (Hunt and Brown, 2005), using polyclonal IgG antibodies which recognize the Nterminus of CD95 (ab13550; Abcam). Primary antibodies were detected using Alexa Fluor 488-or TRITC-conjugated secondary antibodies (Molecular Probes, Eugene OR).…”

“…The cells were cultured as described (Hunt and Brown, 2005) and passaged without attempts to transform or immortalize them. The cells fully maintain their differentiated state when cultured in vitro (Bayreuther et al, 1991;Bradley et al, 1980).…”